Field of the Invention
The present invention relates generally to determining male fertility by measuring the quantity of specific analytes in samples of seminal fluid and more specifically to kits for detecting the concentration of a2V-ATPase, G-CSF, MIP 1α, MCP-1 in such samples.
Background of the Invention
The vacuolar (H+)-ATPase (V-ATPase) is a multi subunit enzyme that couples ATP hydrolysis to the pumping of protons across plasma membranes. It is ubiquitously expressed in eukaryotic cells, where it participates in the acidification of highly differentiated organelles, including the Golgi apparatus, lysosomes, endosomes, and secretory vesicles. In addition, the V-ATPase is also found at high density in the plasma membrane of specialized epithelial cells that are involved in active proton transport and pH regulation of extracellular compartments. Those plasma membrane V-ATPases have important roles in such processes as renal acidification, bone resorption or sperm capacitation. In a murine study, V-ATPases in the apical membrane of epididymal clear cells, which are also controlled by reversible endocytosis and exocytosis, are required for sperm maturation, viability and pH homeostasis. In addition, the a2 isoform of V-ATPase (Atp6v0a2) is located specifically in the acrosomal membrane of murine sperm to regulate an acidic intra-acrosomal pH, which is necessary for processing protease zymogen, essential for fertilization. In agreement with this previous study, Atp6v0a2 was highly expressed in the acrosomal region of the capacitated murine sperm but not detected in non-capacitated sperm from the caudal epididymis. This study provided a new insight into a possible association with Atp6v0a2 and fertilizing ability of capacitated human sperm, since capacitation is required for fertilization and embryogenesis.
Although seminal fluid has been conventionally viewed as transport media for spermatozoa traversing the female reproductive tissues, it is now known to have broader biological actions in regulating female fertility. Seminal fluid contains a complex array of cytokines, chemokines, and other bioactive molecules. Seminal fluid induces pro-inflammatory cytokines and chemokines such as GM-CSF, IL-6, IL-8, MCP-1, MIP-3α, and IL-1α in the female reproductive tract. Particularly, IL-1 has a potential role in the regulation of blastocyst implantation during early pregnancy. IL-1 enhances V-ATPase activity, and increased level of IL-1 may feed back to down regulate the innate immune response, which is essential for implantation. We have shown that Atp6v0a2 can regulate IL-1β as well as IL-1α with little or no subsequent increase in TNF-α secretion. In addition, capacitation appears to cause the release of a2NTD, which is the N-terminal portion from Atp6v0a2. We have shown that a2NTD induces maternal inflammatory cytokines such as LIF, IL-1β, TNF-α and MIP-1α, and exposure of the uterus to sperm accompanied by seminal fluid enhances pregnancy success rate. Therefore, Atp6v0a2 derived from capacitated sperm may play a key role in expression of cytokines and chemokines in the uterus and placenta and controls early inflammatory process which is necessary for implantation and placentation.
In approximately 30% of couples, male factor infertility is the only cause of infertility, and in another 20% to 30% of couples, it is a contributing factor for their infertility. Semen analysis is the most commonly used diagnostic tool for male infertility. Recently, the World Health Organization (WHO) has issued standards for abnormal semen analysis in 2010 (Cooper). However, these standards are not quantitative and do not identify abnormal parameters related to the underlying causes of infertility. To issue these standards semen obtained only from fertile men were used, and there were no “threshold values” for sperm concentration, motility, and morphology to differentiate men as subfertile, of indeterminate fertility, or fertile. Thus, none of these parameters can predict the fertile capacity of sperm or pregnancy outcome with a great deal of confidence. Unfortunately, most clinical laboratories still rely on semen analysis only based on standards to determine plan of care. Indeed, even with techniques such as IVF or IVF with intracytoplasmic sperm injection (ICSI), pregnancy success rates are still remain at 25-30%. This could be partly related to lack of our understanding of the molecular pathology of sperm and semen. Therefore, if a new biomarker could be associated with sperm from infertile men, this would provide a better method to predict fertilization capacity of sperm and pregnancy outcome.
Based on the findings from our lab and others, we hypothesize that Atp6v0a2 in human sperm contributes to the establishment of “immunological privilege”. In this study, we investigated Atp6v0a2 expression and localization in human sperm, and examined the possibility of Atp6v0a2 as a useful biomarker for male factor infertility.
These and other aspects and attributes of the present invention will be discussed with reference to the following drawings and accompanying specification.